Diffuse intrinsic pontine glioma (DIPG) is an aggressive primary brain tumor found exclusively in children. The median survival for DIPG patients is about one year from diagnosis, with no treatment in sight. Recent studies have uncovered driver mutations for DIPG, specifically, somatic mutations in the H3F3A gene have been observed in majority of DIPG cases. H3F3A encodes histone H3 variant H3.3 that plays an important role in regulating gene expression during development. The predominant mutation at H3F3A leads to amino acid change at lysine (K) 27 residue of H3.3 to methionine (M) (H3K27M). In human cells, there are two genes that encode H3.3, which is assembled into nucleosomes in a replication-independent nucleosome assembly pathway. In addition, there are 13 genes encoding canonical histones H3.1 and H3.2, which differ from H3.3 by 4 or five amino acids and are assembled into nucleosomes in a replication-coupled process. Lysine 27 is conserved among all these histone H3 proteins. Therefore, it is unknown how H3.3 K27M mutation, which occurs at one allele of H3F3A gene, promotes tumorigenesis. H3K27 is modified post-translationally by either acetylation or methylation. H3K27 methylation is catalyzed by the PRC2 lysine methyltransferase and plays an important role in gene silencing during stem cell differentiation and maintenance. We and others have shown that a global loss of H3K27 methylation occurs in DIPG cells containing K27M mutation, which is due to inhibition of PRC2 enzymatic activity by H3.3M27 mutant proteins. In addition to the global loss of H3K27me3 that is associated with gene activation, we also observed that H3K27me3 is present at the gene promoters of about 800 genes. These genes are largely silenced and are enriched in pathways associated with tumorigenesis. In addition, we performed an shRNA screen to identify genes that when depleted inhibit the proliferation of DIPG cells. We found that Wnt5a, a protein involved in non-canonical Wnt signaling pathway, inhibits proliferation of DIPG cells. Based on these exciting results, we hypothesize that H3.3M27 mutation proteins reprogram epigenetic states and gene expression of a key regulator(s)/pathway(s) and thereby promotes tumorigenesis. In this proposal, we will elucidate the molecular mechanism whereby the 800 genes with H3K27me3 and determine to what extent ?gain? of H3K27me3 at these genes promotes tumorigenesis. In addition, we will elucidate the molecular mechanisms whereby Wnt5a and several components of Wnt5a signaling pathway are required for the proliferation of DIPG cells and test the hypothesis that inhibition of components of Wnt5a signaling pathway is a viable approach for the future treatment of DIPG. Together, the proposed studies will not only discover the molecular basis of tumorigenesis of DIPG, but also lay the foundation for molecularly targeted therapies for this deadly disease.